A hospital based pilot study on Epstein-Barr virus in suspected infectious mononucleosis pediatric patients in India

Introduction: Infectious mononucleosis (IM) caused by the Epstein-Barr virus (EBV) is commonly diagnosed by detection of antibodies in the patient’s sera. Differentiation of acute from chronic and differential diagnosis of EBV-induced IM from IM-like syndrome caused by human cytomegalovirus (CMV) is important. The objective of this study was to standardize and use polymerase chain reaction (PCR) for diagnosis of EBV and evaluate it against enzyme-linked immunosorbent assay (ELISA). Methodology: ELISA for detection of IgM and IgG antibodies to viral capsid antigen (VCA) and PCR targeting the VCA and EBNA1 gene of EBV and mtrII gene of CMV were performed on180 peripheral blood samples collected from 180 patients with suspected IM. The analytical sensitivity of PCR was evaluated against that of ELISA. Results: Using the standard serological profile as the reference, the EBV-VCA gene was detected in 41 (95%) of 45 samples collected from patients with early primary infections, in 41 (54%) of 75 with recent primary infections, and in7 (17%) of 39 with past infections. The result of VCA PCR was statistically significant in virus detection during early or primary stage of infection. Nineteen (49%) EBV-seropositive samples were positive for CMV by PCR. All control samples tested negative for both VCA and EBNA1by PCR. Conclusions: VCA PCR is sensitive for the detection of EBV DNA in the early or primary stage of infection and can be considered a reliable method to rule out the cross-reactivity and differential diagnosis of EBV-induced IM from IM-like syndrome.


Introduction
Infectious mononucleosis caused by the Epstein-Barr virus is one of the most common infections reported among children and adults, followed by human cytomegalovirus.In India, very few publications are available on EBV-associated IM.The Epstein-Barr virus is contagious, as it can be contracted through direct contact with an infected person's saliva.About 95% of the population has been exposed to this virus by the age of 40, but only 15%-20% of teenagers and about 40% of those adults exposed to the virus become infected [1].
Antiviral agents, namely acyclovir or valacyclovir, are recommended to treat children with severe infection or those who develop complications [2].It has also been reported that patients with lymphoma and leukemia may present with clinical symptoms suggestive of infectious mononucleosis [3].The most commonly used diagnostic criterion is demonstration of atypical lymphocytes in peripheral blood smear.However, presence of atypical lymphocytes alone is not sufficient for confirmation of mononucleosis.Further serological confirmation by demonstrating the presence of IgG and IgM antibodies produced against different EBV antigens/CMV is essential [4].When IgM class of antibody to viral capsid antigen (VCA) alone are detected, an early primary infection is suspected; when IgG class of antibody to VCA alone are detected, a past infection is suspected; whereas, when both IgM and IgG positivity occurs, a recent infection or reactivation is suspected [5].During early primary infection, IgM class of antibody to Epstein Barr viral capsid antigen is detected, whereas detection of IgG to VCA is seen in past infections and both IgM and IgG antibodies are detected during recent infection or reactivation.
Although serological investigations are preferred for diagnosis of CMV/EBV infection where the result of EBV serology presents a high degree of variability, serology provides rational criteria for interpretation of the results [6].The detection of antibodies is less useful in immunocompromised patients because of their immune system dysfunctions, and because the type of antibody and its maintenance may vary over time depending on the dynamics of the disease, thus leading to atypical profiles [7].
In the current study, polymerase chain reaction (PCR) targeting VCA designed by us and PCR targeting EBNA were evaluated against serological methods in the diagnosis of IM caused by EBV.The comparative applicability of both the methodologies to distinguish acute and latent EBV infection was determined.A correlative analysis of laboratory diagnosis and clinical presentations was done.

Methodology
The study was conducted at L& T Microbiology Research Centre, Vision Research Foundation, Chennai, after approval from the institutional research and ethical board (IRB) was obtained.

Patients and clinical specimens
Sixty peripheral blood samples were collected from immunocompetent healthy volunteer donors and served as controls.Donors were between 17 and 20 years of age.A total of 180 peripheral blood samples were collected between August 2010 and July 2013 from180 immunocompetent patients clinically suspected to have infectious mononucleosis at a tertiary child care center in Chennai.The ages of the patients ranged from 2.5 months to 14 years.All patients had EBV-related symptoms, namely fever, rash, lymphadenopathy, pharyngitis, hepatomegaly, or splenomegaly.
Children with EBV-associated malignant diseases such as malignant lymphoma or chronic active infections were excluded.
Samples were collected after getting the informed consent from the patient or patient'sguardian.Clinical details were recorded by the clinician in the proforma made specifically for the study.
Two to three milliliters of the peripheral blood were collected in EDTA anticoagulated vacutainers and transported to a cold environment within four hours of collection.At the laboratory, the specimens were processed under sterile conditions for separation of plasma from the EDTA vacutainer, followed by separation of Buffy coat.The specimens were stored at -20°C.In addition, 2 mL of blood was collected in a vacutainer without any anticoagulant.Serum samples were separated and stored at 4°C until they were assayed.The serological and PCR tests were carried out in batches.

Enzyme immunoassay (EIA)
Human IgG and IgM antibodies against EBV-VCA were tested by EIA following the manufacturer's instructions (Demeditec Diagnostics, Kiel, Germany).All the samples were tested in duplicate.The test was also carried out in duplicate.The patients who had detectable IgM antibodies to VCA and an absence of VCA-IgG were considered to have early primary infection.Past infection was defined as a positive assay for IgG to VCA and negative for IgM to VCA.Recent infection or reactivation was defined as a positive assay for both IgM and IgG to VCA, and a negative assay for both IgM and IgG was defined as no EBV infection [5].

DNA extraction
Leucocytes of the Buffy coat suspended in 100μL of plasma were subjected to DNA extraction following the manufacturer's instructions (QIAGEN DNA extraction kit, Hilden, Germany).The extracted DNA was amplified for the detection of genes coding for VCA and EBNA1.

Semi-nested amplification of the EBV-VCA gene
The semi-nested PCR for detection of the EBV-VCA gene was standardized using the EBV standard strain culture infiltrate of marmoset cell line infected with EBV B958 (National Eye Institute, Bethesda, USA).Briefly, 10 μL of positive control DNA elute was subjected to amplification of the EBV-VCA gene.Primers targeting the VCA gene were designed using Primer Premier (Premier Biosoft International, Palo Alto, USA) based on consensus sequence obtained with VCA sequences of EBV submitted in GenBank.The nucleotide sequences of the primers and the expected respective product size are shown in Table 1.The nucleotide location of the first-round primers on the genome is 77002-77174, and that of second-round primers is 77058-77174.All primers and PCR reagents were procured from VBC Biotech Service (Vienna, Austria).

Optimization of VCA PCR
Both VCA and EBNA1 PCR were optimized to be carried out in the same thermal profile through gradient PCR temperature profile.The PCR mixture (50 μL) contained 100 mM of dNTP mixture, 10X PCR buffer with 15mM MgCl 2 , 1 μM of each forward and reverse primer, and 3U/μL Taq DNA polymerase.Ten microliters of extracted positive control DNA was added to the first-round PCR reaction mixture.The reaction mix was incubated in the thermal cycler as follows: denaturing the DNA at 94°C for 5 minutes followed by amplification for 30 cycles, secondary denaturation at 94°C for 1 minute, annealing at 59°C for 1 minute and extension at 72°C for 1 minute, with final extension for 7 minutes at72°C.For the secondround amplification, 5μL of the first round product was added to 50μL of the PCR mix containing 10 mM of each dNTP, 10X buffer, 1 μM of each forward and reverse primer, and Taq DNA polymerase.The PCR amplification was carried out for 20 cycles with the same thermal profile as mentioned above.PCR products were analyzed by2% agarose gel electrophoresis.Two controls (one reagent control and one reaction control) were included in each PCR run.The PCR results were considered valid only when the reagent controls were negative and the specific amplified product was obtained with the positive controls.To prevent contamination ofDNA extraction, PCR cocktail preparation, amplification, and analysis of results were carried out in physically separated rooms.Visualization of PCR product was done by subjecting 10 μL of amplified reaction mixture to electrophoresis on a 2% agarose gel incorporating 5 μg mL-1 of ethidium bromide in 1X Tris-Borate buffer (pH -8.2-8.6) and was documented on a gel documentation system (VilberLourmat, France).

Sensitivity of PCR in detecting the EBV-VCA gene
DNA was extracted from 200 µL of EBV standard strain culture infiltrate of marmoset cell line infected with EBV B958 (National Eye Institute, Bethesda, USA); the concentration was primarily quantified using nanovue (GE Healthcare, Chennai, India).Serial tenfold dilutions of the DNA were made from 10 -1 to 10 -10 (i.e., 5µL of DNA with 45µL of Milli-Q water).From each dilution, 10L of DNA was taken for PCR reaction.The lowest dilution showing PCR positivity and its corresponding DNA concentration was calculated and considered as the sensitivity of the PCR.

Nested amplification of the EBNA1 gene [8]
Nested PCR was optimized and performed for all the samples collected targeting Epstein-Barr nuclear antigen 1(EBNA1).The primers used for the nested PCR amplification are listed in table 1.

Nested amplification of the mtrII gene of CMV [9]
Nested PCR for detection of CMV was performed on all samples collected targeting the mtrII gene of CMV.The primers used for nested PCR amplification are listed in Table 1.

Statistical methods
All the statistical analyses were carried out using Statistical Package for Social Sciences (SPSS) software version 14.0.Clinical sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and P value of VCA PCR were determined.A p value < 0.05 was accepted as statistically significant.were categorized into group B (n=20).Group C (n=15) consisted of samples that tested positive for the IgG class of antibodies to VCA and negative for both PCRs.Group D (n=21) included samples that were both seronegative and PCR negative for detection of EBV.
Four samples belonging to group A tested IgG positive for CMV ELISA and were also found to have CMV DNA by PCR.Both CMV ELISA and PCR were negative in20other clinical samples belonging to group B. CMV PCR and both IgM and IgG antibodies against CMV were positive in samples belonging to group C. Twenty-one other samples categorized under group D tested negative for CMV by both ELISA and PCR (table 3).The CMV PCR results were reproducible in all samples tested positive and negative.

Clinical signs and symptoms
All the 180 patients included in the study were suspected to have infectious mononucleosis with presentation of high-grade fever and cough, and all the patients included had shown one of the criteria of infectious mononucleosis, namely lymphocytosis or the presence of atypical lymphocyte in the blood smear study.Among the 180 patients, 45 seropositive (VCA IgM positive) patients presented with decreased oral intake, pharyngitis lymph adenopathy, hepatosplenomegaly, abnormal liver function test, and/or rhinorrhea, in addition to fever and cough.The most common clinical symptoms observed among patients with early primary infection were high-grade fever and decreased oral intake.

Discussion
Though serological tests are considered a reliable tool by clinicians in the diagnosis of infectious mononucleosis caused by EBV, these tests do have demerits.IgM titer declines or disappears within four weeks of infection.EBV infection can be mistaken for CMV, streptococcal, or throat infection or an ordinary fever, strictly because of the symptom overlap.
Chan et al. concluded that neither a test of EBV VCA IgM nor a test of the presence of VCA IgG in the absence of EBNA1 antibody is reliable for diagnosing primary EBV infection.PCR for EBV DNA in plasma or serum is a useful addition to the panel of tests available for this purpose, particularly if used as a confirmatory test in conjunction with serological tests [10].
Using the standard serological profile as the referenced gold standard, the EBV-VCA gene was detectable in 41 (95%) of 45 patients diagnosed with clinical symptoms suggestive of early primary infections, 41 (54%) of 75 with recent primary infections, 7 (17%) of 39 with past infections, and none (0%) of 21 with no infection.Based on our study, it can be concluded that the PCR remains negative in seropositive patients and seropositive healthy donors in the absence of active EBV infection.The EBV/CMV cross-reaction as the reason for falsepositive EBV-VCA IgM reactions was confirmed by performing PCR targeting the mtrII gene of CMV.CMV was detected by PCR in four samples found positive for EBV IgM antibodies and negative for EBV VCA PCR .Four EBV seropositive samples in which IgM antibodies to VCA were detected and EBV-VCA PCR was negative, were tested positive for CMV PCR.Therefore, in the current study, the standardized VCA PCR helped to detect EBV false IgM seropositive results.
The comparison of clinical data of patients diagnosed with early primary EBV infection based on PCR assay and serology revealed that high-grade fever and cough were observed in all groups.The study patients' symptoms included decreased oral intake, pharyngitis, lymph adenopathy, hepatosplenomegaly, abnormal or altered liver function, and rhinorrhea; however, these symptoms were observed only among the 45 seropositive (VCA IgM positive) patients(with early/primary infection)in addition to fever and cough observed in other patients.Forty-one among these 45 patients were positive for VCA PCR.No significant correlation of clinical symptoms was observed in the rest of the population, signifying that VCA PCR is highly sensitive and specific for the differential detection of EBV DNA in the early or primary stage of infection and can be considered a reliable method to rule out the cross-reactivity and differential diagnosis of EBV-induced infectious mononucleosis from infectious mononucleosis-like syndrome.
The limitations of the present study are mainly related to the study group.If the samples collected from both controls and patients belonged to same age group, the results obtained would better help to explain the seroprevalence among a healthy population when compared with an infected population.The tests should be done on follow-up samples to further understand the mechanism of infection and disease.
The merit of this study is that the standardized PCR targeting EBV helped in the early diagnosis of infection caused by EBV and this study proved that PCR could differentially diagnose the infectious agents in patients having antibodies to both EBV and CMV.

Conclusions
VCA PCR is sensitive for the detection of EBV DNA in the early or primary stage of infection and can be considered a reliable method to rule out the crossreactivity and differential diagnosis of EBV-induced IM from IM-like syndrome.

Table 1 .
List of primers used for amplification of genes that code for VCA, EBNA1 of EBV and mtrII gene of CMV VCA: viral capsid antigen; EBV: Epstein-Barr virus